HYDROPHOBIC AMINO-ACID-RESIDUES OF HUMAN ANTICOAGULATION PROTEIN-C THAT CONTRIBUTE TO ITS FUNCTIONAL BINDING TO PHOSPHOLIPID-VESICLES

The contributions to functional phospholipid (PL) binding of the clust er of amino acid side chains of human protein C (PC) comprising F-4, L (5), and L(8) have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain re placed the wild-type hydrophobic groups at these positions. The PL-dep endent plasma-based anticoagulant activities of [F(4)Q]r-APC and [L(8) Q]r-APC were severely reduced to 5% and <2%, respectively, of wild-typ e r-APC. Activity losses of the mutants toward inactivation of coagula tion factor VIII, measured in the complete in vitro tenase system, hav e also been observed. As evidenced through Ca2+-induced intrinsic fluo rescence changes, both [F(4)Q]r-PC and [L(8)Q]r-PC were able to adopt Ca2+-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca2+-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L(8)Q]r-PC showed greatly defective macroscopic binding proper ties to PL vesicles, as did to a lesser extent [F(4)Q]r-PC. These find ings were similar to those reported previously for [L(5)Q]r-PC/APC [Zh ang, L., and Castellino, F. J. (1994) J. Biol. Chern. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutant s are related to their suboptimal binding to PL or to their misorienta tion on the PL surface leading to poor alignment of the active sites o f the r-APC mutants with the complementary cleavage sites on fVIII/fVI IIa and fV/fVa. These investigations confirm the importance of hydroph obic components of functional PL binding by PC and APC and implicate L (8), along with L(5), as the principal amino acid residue involved in these interactions. F-4 has a lesser direct involvement in this regard but may contribute to proper alignment of PC and APC on the PL surfac e.